2.9.2. In Vitro Hemolysis Assay

This assay was performed according to Memar et al.’s protocol [52]. Briefly, human blood from a healthy volunteer was collected, centrifuged at 664× g for 5 min, and washed with PBS three times.

Two doses of peptide (8 μg and 16 μg) were prepared in 100 μL of PBS (1×). One hundred μL of washed RBCs suspension (2%) was added to each well of a 96-well microplate (Nunc, Sigma Co., St. Louis, MO, USA). Phosphate buffer saline and Triton X-100 (0.1%) was used as negative and positive control, respectively. The microplate was incubated at 37 °C for 2 h and centrifuged at 1664× g for 10 min. The supernatant was transferred to a new plate and OD was measured at 540 nm in a microplate spectrophotometer (EPOCH, BioTek, Winooski, VT, USA). The experiment was carried out in triplicate. The degree of hemolysis was determined as the following formula: